Expression of lipoxygenase during organogenic nodule formation from hop internodes.

Study of lipoxygenase expression (LOX; EC 1.13.11.12) during organogenic nodule formation in hop (Humulus lupulus var. Nugget) showed that LOXs are developmentally regulated throughout the process, suggesting their involvement in the response of internodes to wounding, nodule formation, and plantlet regeneration from these nodules. LOX activity and lipid peroxides exhibited a huge increase during the first week of culture, which may indicate a role for LOX and LOX products in response to wounding in hop, as reported for other systems. Western blotting analysis showed a de novo synthesis of LOX isoenzymes in response to wounding and the detection of three different isoenzymes. Confocal analysis of LOX immunofluorescence revealed the presence of the enzyme in cortical cells of induced internodes and in prenodular cells, mostly appearing as cytoplasmic spots. Some of them were identified as lipid bodies by cytochemical and double immunofluorescence assays, suggesting the involvement of a lipid body LOX during nodule formation. Immunogold labeling detected LOX in peroxisomes, lipid bodies, and plastids of nodular cells. Quantification of the labeling density provided statistical significance for the localization of LOX (three different isoenzymes) in the three compartments, which suggested a possible involvement of LOX in metabolic functions of these organelles during organogenic nodule formation and plantlet regeneration.     

Differential expression and cellular localization of ERKs during organogenic nodule formation from internodes of Humulus lupulus var. Nugget

The expression and subcellular localization of extracellular signal-regulated kinase 1 or 2 (ERK1/2) homologues (HLERK1/2) during the process of organogenic nodule formation in Humulus lupulus var. Nugget was studied using antibodies specific for ERK1 and ERK2, and for phosphorylated mitogen-activated protein kinases (MAPKs). The increase in HLERK levels, detected by Western blotting 12 hours after wounding suggests their involvement in response to the wounding treatment applied for morphogenesis induction. In dividing cambial cells, occurring in between 4 and 7 days after morphogenesis induction, as well as in dividing prenodular cells (15 days after induction) HLERK1 and/or 2 were localized in the nucleus. However, as soon as nodular cells start proliferating to form shoot meristems, HLERK1 and 2 were detected in the cytoplasm and not in the nucleus. The data reported account for a differential expression and activation of HLERK1 and HLERK2 throughout the process of nodule formation and plantlet regeneration. HLERK1 appears to be expressed in the stages of nodule formation and plantlet regeneration, playing a possible role in controlling cell proliferation and differentiation. HLERK2 may be induced as a response to reactive oxygen species (ROS) generated by wounding of internodes as its expression is reduced in liquid medium with less oxygen availability compared to solid medium. However, addition of a ROS inhibitor to the liquid medium does not result in a further decrease in the HLERK2 level.     

Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis(+)12-oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato. In stems, young leaves and young flowers, AOC mRNA accumulates to a low level, contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g-1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue-specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue-specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink-source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin-mediated wound response of tomato.     

Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."     

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.     

Role of cis-12-oxo-phytodienoic acid in tomato embryo development

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Constitutive overexpression of allene oxide cyclase in tomato (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and octadecanoids in flower organs but not in leaves

The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.     

Octadecanoid signaling component "burst" in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan

Octadecanoid pathway components, 12-oxo-phytodieonic acid (OPDA) and jasmonic acid (JA), are key biologically active regulators of plant self-defense response(s). However, to date these compounds have been studied mostly in dicots, and used large (1-10 g fresh weight, FW) samples for quantification, even when examined in mature rice plants, which is a drawback considering their rapid responsiveness to stress. Focusing on rice--a monocot cereal crop research model--this work describes an efficient and simultaneous quantification of both OPDA and JA using a minimum amount of 200mg FW seedling leaf tissue upon wounding (by cut) and treatment with fungal elicitor, chitosan (CT) by high-pressure liquid chromatography-turboionspray tandem mass spectrometry. Transient OPDA/JA "burst" was consistently and reproducibly detected within 3 min in wounded and CT treated leaves. OPDA peaked dramatically around 5 min and returned to its basal level within 15 min, whereas JA induction upon wounding and CT treatment were in parallel to OPDA production, peaking at 30 and 60 min, respectively. Present results mark a major advance in our understanding of key inducible octadecanoid pathway components in rice, and strongly suggest a role for the octadecanoid pathway downstream of perception of at least these two fundamentally different extracellular stimuli.     

Jasmonate biosynthesis in Arabidopsis thaliana requires peroxisomal beta-oxidation enzymes--additional proof by properties of pex6 and aim1

Jasmonic acid (JA) is an important regulator of plant development and stress responses. Several enzymes involved in the biosynthesis of JA from alpha-linolenic acid have been characterized. The final biosynthesis steps are the beta-oxidation of 12-oxo-phytoenoic acid. We analyzed JA biosynthesis in the Arabidopsis mutants pex6, affected in peroxisome biogenesis, and aim1, disrupted in fatty acid beta-oxidation. Upon wounding, these mutants exhibit reduced JA levels compared to wild type. pex6 accumulated the precursor OPDA. Feeding experiments with deuterated OPDA substantiate this accumulation pattern, suggesting the mutants are impaired in the beta-oxidation of JA biosynthesis at different steps. Decreased expression of JA-responsive genes, such as VSP1, VSP2, AtJRG21 and LOX2, following wounding in the mutants compared to the wild type reflects the reduced JA levels of the mutants. By use of these additional mutants in combination with feeding experiments, the necessity of functional peroxisomes for JA-biosynthesis is confirmed. Furthermore an essential function of one of the two multifunctional proteins of fatty acid beta-oxidation (AIM1) for wound-induced JA formation is demonstrated for the first time. These data confirm that JA biosynthesis occurs via peroxisomal fatty acid beta-oxidation machinery.     

Occurrence of the allene oxide cyclase in different organs and tissues of Arabidopsis thaliana

Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.     

Induction of the Arabidopsis PHO1;H10 gene by 12-oxo-phytodienoic acid but not jasmonic acid via a CORONATINE INSENSITIVE1-dependent pathway

Expression of AtPHO1;H10, a member of the Arabidopsis (Arabidopsis thaliana) PHO1 gene family, is strongly induced following numerous abiotic and biotic stresses, including wounding, dehydration, cold, salt, and pathogen attack. AtPHO1;H10 expression by wounding was localized to the cells in the close vicinity of the wound site. AtPHO1;H10 expression was increased by application of the jasmonic acid (JA) precursor 12-oxo-phytodienoic acid (OPDA), but not by JA or coronatine. Surprisingly, induction of AtPHO1;H10 by OPDA was dependent on the presence of CORONATINE INSENSITIVE1 (COI1). The induction of AtPHO1;H10 expression by wounding and dehydration was dependent on COI1 and was comparable in both the wild type and the OPDA reductase 3-deficient (opr3) mutant. In contrast, induction of AtPHO1;H10 expression by exogenous abscisic acid (ABA) was independent of the presence of either OPDA or COI1, but was strongly decreased in the ABA-insensitive mutant abi1-1. The involvement of the ABA pathway in regulating AtPHO1;H10 was distinct between wounding and dehydration, with induction of AtPHO1;H10 by wounding being comparable to wild type in the ABA-deficient mutant aba1-3 and abi1-1, whereas a strong reduction in AtPHO1;H10 expression occurred in aba1-3 and abi1-1 following dehydration. Together, these results reveal that OPDA can modulate gene expression via COI1 in a manner distinct from JA, and independently from ABA. Furthermore, the implication of the ABA pathway in coregulating AtPHO1;H10 expression is dependent on the abiotic stress applied, being weak under wounding but strong upon dehydration.     

Jasmonate biosynthesis in legume and actinorhizal nodules

Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells. The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild-type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance. To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1-sense or MtAOC1-RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation. These data suggest that jasmonates are not involved in the development and function of root nodules.